Corneal endothelial cells are different from vascular and pulmonary xe2x80x9cendothelial cellsxe2x80x9d as they have a different embryonic tissue origin. Human corneal endothelial cells do not normally proliferate in vivo to replace those lost due to cell injury or death. Growth of these cells in culture is also extremely limited. This can be a serious problem as age, diseases such as glaucoma and diabetes, and ocular surgical procedures, such as laser vision correction and cataract extraction and intraocular lens implantation, cause an accelerated loss of these precious cells. There are no medical treatments for corneal diseases resulting from endothelial cell loss. Currently, corneal transplantation is the only way to restore normal vision.
The relative ability of corneal endothelial cells to proliferate in vivo and in culture appears to be a function of age; i.e., embryonic corneal endothelial cells and cells from neonates will proliferate much more readily than cells from children and adults. In a few cases, researchers have been able to culture cells from older donors, but growth has been supported by seeding the cells onto an artificial matrix, such as chondroitin sulfate/laminin, or onto extracellular matrix secreted by corneal endothelial cells from cows, one of a number of species whose corneal endothelial cells do grow readily in culture. A reliable way of growing and/or supporting human corneal endothelial cells, whether in vitro or in vivo, would be highly desirable. In particular, there remains a need for stimulating proliferation of adult corneal endothelial cells.
The invention provides a new source of corneal endothelial cells for use in research and treatment of injured or diseased corneal endothelium. In particular, the invention enables the growth/proliferation of human corneal endothelial cells from adult humans, preferably those aged fifty or more years.
One aspect provides a simple, yet effective method of stimulating or promoting the proliferation of adult corneal endothelial cells, whether in vitro or in vivo. The invention encompasses exposing corneal endothelial cells to an agent that interrupts cell-to-cell contact, also called xe2x80x9ccell-cell adhesionxe2x80x9d or xe2x80x9ccell-cell contactxe2x80x9d. Such cell-cell contact interruption step preferably is sandwiched between two incubation or exposure steps, in which adult-derived corneal endothelial cells are exposed to at least one growth factor, optionally in a growth medium. xe2x80x9cGrowth factorxe2x80x9d refers to any factor that induces a corneal endothelial cell to enter the cell cycle and thus to proliferate. Exemplary growth factors include, but are not limited to, e.g., one or more mitogen(s) such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and/or nerve growth factor (NGF). Other growth factors effective for corneal endothelial proliferation are known in the art and may be used in the invention.
The agent used to interrupt cell-cell contacts may be a calcium chelator such as ethylenediaminetetraacetic acid (EDTA), a known chelator of calcium and magnesium ions previously used in many laboratories to release cultured cells from contact inhibition. When added to a corneal culture medium, EDTA released cells from contact inhibition and promoted proliferation in corneal endothelium from older donors. Based on the observations reported here, it is expected that corneal endothelium from older individuals will divide in situ by exposing them to positive growth factors under conditions in which cells have been transiently released from contact inhibition. Human corneal endothelial cell density decreases with age (1,2), indicating that these cells do not replicate sufficiently to replace dead or injured cells. Transfection studies using viral oncoproteins have demonstrated that human corneal endothelium has an intrinsic high proliferative capacity (3,4). The fact that endothelial cells possess the capability to divide, but normally do not replicate in vivo, suggests that they are actively maintained in a non-replicative state. Cell cycle studies indicate that human corneal endothelial cells in vivo are arrested in the G1-phase of the cell cycle (5,6). Among the factors that may be responsible for maintaining these cells in G1-phase arrest, are a lack of available positive growth factors (7) and the presence in aqueous humor of transforming growth factor-xcex22 (TGF-xcex22), which has been found to suppress S-phase entry in cultured corneal endothelial cells (8,9). In addition, studies of the developing cornea in neonatal rats suggest that a contact inhibition-like mechanism may actively suppress replication in the mature endothelial monolayer (10). Other evidence for cell contact-mediated regulation of proliferation is that corneal endothelial cells will divide in response to wounding (11,12). In tissue culture and in organ cultured corneas, only endothelial cells adjacent to the wound edge or cells that have migrated into the wound bed will proliferate, demonstrating the importance of releasing cell-cell contacts in order to promote proliferation. Formation of cell-cell contacts or adhesions is mediated by a number of proteins that are associated with different types of junctional complexes, including cadherins (13) (adhering junctions), ZO-1 (14) (tight junctions), and connexin-43 (15) (gap junctions). These proteins all require calcium for maintenance of their adhesion function. In low-calcium environments, junctional complexes mediated by these proteins disassemble and cell-cell contact is broken. Exposure of the corneal endothelium to calcium-free medium causes disruption of apical junctional complexes, increased transendothelial perfusion, and corneal edema (16-18). These changes can be reversed by replacing calcium in the medium (17,18), or by exposing cells to ionophores that release intracellular calcium stores(18).